1. Field of the Invention
The present invention relates to 4-Pi microscopes.
2. Description of the Related Art
(Note: This application references a number of different publications as indicated throughout the specification by one or more reference numbers within brackets, e.g., [x]. A list of these different publications ordered according to these reference numbers can be found below in the section entitled “References.” Each of these publications is incorporated by reference herein.)
In this section, the present invention explains the concept and importance of 4pi fluorescence microscopy.
Microscopy is one of the most important tools in biomedical research. Over the past few decades, advanced optical imaging technologies have significantly expanded the capability of optical microscopy, providing high spatial resolution and molecular information. Among the various techniques, fluorescence microcopy provides excellent sensitivity and has gained its popularity among biologists since the introduction of a green fluorescence protein (GFP) [1, 2].
For three dimensional (3D) high resolution imaging, confocal microscopy is often used. With a pinhole at the focal plane rejecting the out-of-focus light, confocal microscopy can provide 3D images with ˜600 nm axial resolution and ˜200 nm lateral resolution.
The difference between the axial and the lateral resolution is associated to the angular distribution of the optical field of a single objective lens. FIG. 1 (a) shows the light intensity distribution at the focus of a Numerical Aperture (NA) 1.3 oil immersion objective lens illuminated by a collimated laser beam (λ=532 nm, filling factor=1). Even with such a high NA objective, the focus is still axially elongated, which can be understood by noting that the wave vectors of a single objective lens have components propagating to the left and the right along the lateral axis, but only a single direction (up or down) along the axial axis. The axial resolution can be significantly increased by adding a properly aligned opposing lens. The coherent addition of a second focus can create smaller excitation spots as shown in FIG. 1 (b). The imaging method employing two coherent opposing objective lenses in confocal fluorescence microscopy is known as 4Pi microscopy [3].
The resolution of confocal microscopy (including 4Pi) is determined by the product of excitation point spread function (PSF) and detection PSF, which gives rise to three different 4Pi systems (type A, B, and C) [3, 4].
In a type A 4Pi system, two excitation laser beams enter both objective lenses and the fluorescence signal exiting from one lens is used for confocal detection.
In a type B system, a single excitation beam enters one objective lens and the fluorescence signals exiting from both lenses are brought into coherent interference at the confocal detection system. Fluorescence emission is incoherent in that the emitted photons are uncorrelated. The interference effect employed in type B system is not between different photons but between the two different paths of a single emitted photon. Such an effect has also been employed for fluorescence OCT (optical coherence tomography) systems [5].
Type A and B systems can achieve similar spatial resolutions (axial ˜120 nm, lateral ˜200 nm). A further improvement on axial resolution can be made by combining type A and B systems such that two excitation beams enter both objective lenses and the fluorescence signals exiting both lenses are brought to interference at the confocal detection system, which is known as type C 4Pi system.
Despite the simple concept, the implementation of 4Pi microscope requires significant expertise in optics. Type A 4Pi may be used as an example to discuss some of the difficulties. First, to establish good interference between the two foci, the two opposing high NA (numerical aperture) oil immersion lenses need to be precisely aligned since inaccurate alignment can reduce the interference fringe contrast and lower the image quality. Second, the phase difference between the two excitation beams needs to be stabilized during the imaging process.
As can been see from FIG. 1 (c), the axial distribution of a 4Pi system comprises three peaks of the same width above the half maximum of intensity which means a single isolated fluorophore generates three images in an axial scan. The side peaks can be suppressed by multiplying the excitation PSF with the 4Pi detection PSF (type C 4Pi), by implementing two-photon excitation, and by using higher NA objectives such as Nikon Apo TIRF 60× NA 1.49 oil. The residue side peaks can be further removed mathematically with deconvolution algorithms. If the relative phase between the two excitation beams varies, the excitation PSF will be modified.
FIG. 1 (d) shows the scenario when the relative phase is changed by π. As expected, the peaks and valleys interchange as compared to FIG. 1 (b) and there are four peaks above half maximum as shown in FIG. 1 (e). If such a phase variation happens during imaging, the acquired image cannot be deconvoluted with a single axial distribution profile any more, resulting in image distortion. Experimentally, a compact and enclosed system is required to minimize the phase drift due to air current and mechanical perturbation, and phase stabilizing devices such as closed-loop piezo mirrors are often employed to improve the phase stability. Third, the imaged sample needs to be thin and optically homogeneous. Inhomogeneous region can cause aberration and distort the focus, altering the PSF.
Embodiments of the present invention use Optical Phase Conjugation (OPC) systems to solve the first (accurate alignment) and the third difficulties (optical aberration) of 4Pi microscopy. Specifically, a system is designed that is capable of automatic focus alignment and aberration compensation that permits using 4Pi microscopy for complex biological samples as embryos.